DNA sense-and-respond protein modules for mammalian cells
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Citation Slomovic, Shimyn and Collins, James J. “DNA Sense-and-Respond
Protein Modules for Mammalian Cells.” Nature Methods 12, no. 11 (September 21, 2015): 1085–1090. © 2015 Macmillan Publishers Limited, part of Springer Nature
As Published http://dx.doi.org/10.1038/nmeth.3585
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Citable link http://hdl.handle.net/1721.1/108502
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DNA sense-‐and-‐respond protein modules for mammalian cells
Shimyn Slomovic1,2,3 and James J. Collins1-‐6*
1. Institute for Medical Engineering & Science, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
2. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. 3. Synthetic Biology Center, MIT, Cambridge, Massachusetts, USA.
4. Harvard-‐MIT Program in Health Sciences and Technology
5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
6. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
* email: jimjc@mit.edu phone: 617-‐324-‐6607 fax: 617-‐253-‐7498
We have generated synthetic protein components that can detect specific DNA sequences and subsequently trigger a desired intracellular response. These modular sensors exploit the programmability of zinc finger DNA-‐recognition to drive the intein-‐mediated splicing of an artificial trans-‐activator that signals to a genetic circuit containing a given reporter or response gene. We utilize the sensors to mediate sequence recognition-‐induced apoptosis, as well as to detect and report a viral infection. This work establishes a synthetic biology framework for endowing mammalian cells with sentinel capabilities, providing a programmable means to cull infected cells, and for identifying positively transduced or transfected cells, isolating recipients of intentional genomic edits, and increasing the repertoire of inducible parts in synthetic biology.
Cys2His2 ZFs zinc finger proteins (ZFs) have long been recognized for their potential
as artificial DNA binding proteins. Various methods, such as partially randomized libraries or single-‐finger modular assembly1,2, may be used to design ZFs to bind any
DNA sequence, enabling a range of applications. For example, when attached to the
Fok I endonucleolytic domain, ZF pairs can facilitate genomic editing3. Through
SEquence Enabled Reassembly (SEER), ZFs joined to split b-‐lactamase can function as in vitro diagnostic tools4. Also, fusion to the viral trans-‐activating domain, VP64,
generated synthetic transcription factors (TFs) that drive engineered regulatory circuits in yeast or mammalian cells, furthering our understanding of transcriptional cooperativity in eukaryotes5, 6. Additionally, ZFs fused to chromatin regulators have
been used to investigate epigenetics, revealing synergistic activation and spatial regulation7.
Commonly, whether utilizing ZFs or other genome engineering tools, such as TALEs or CRISPR-‐Cas, the tethered, delivered activity acts locally on the DNA target itself, e.g., endonucleolytic cleavage or transcriptional modulation8. There are
however instances in which it is desirable that the detection of a specific
endogenous or synthetic DNA sequence trigger a tailored response. Responses could include an amplified report signal indicating provirus presence, a chromosomal transposition, or an intentional genomic alteration. Others might be immune system recruitment to the site of infection or tumor, or kill-‐switch activation that induces apoptosis to prevent pathogen spread. Accordingly, we sought to forward engineer a system that could utilize the programmable nature of ZFs for target detection and deliver the recognition signal to an easily modifiable response circuit.
To bridge the gap between DNA recognition and trans-‐response, we used inteins; peptides that splice their flanking regions, referred to as exteins9,10. In
pioneering work on Conditional Protein Splicing (CPS), the contiguous
Saccharomyces cerevisiae SceVMA intein was artificially split, rendering the separate halves inactive such that they require a condition for co-‐localization and reassembly
11. After fusing one member of the rapamycin-‐binding heterodimeric pair, FRB-‐
FKBP, to each of the split SceVMA intein-‐extein halves, rapamycin served as a condition for their co-‐localization12. The repertoire of trans-‐splice-‐inducing
conditions has expanded and now includes temperature, light, and protein scaffold activation13,14,15. DNA-‐recognition can also induce splicing, as shown in a previous
study wherein ZFs were fused to a split intein/luciferase construct and their binding signal measured drug-‐induced DNA demethylation16.
Here, we sought to use DNA-‐sensing modules as dimerizing domains (Fig. 1). Programming two ZF-‐based sensors to bind adjacently within the targeted DNA sequence would allow for conditional intein co-‐localization and trans-‐splicing of a response factor that activates any gene installed in a response circuit. Below, we describe the engineering of a modular sequence-‐recognition system capable of producing a customizable response signal, and demonstrate its ability to mediate sequence recognition-‐induced apoptosis and virus detection in infected, human cells.
Results
At the onset of this work, we utilized a library of ten tridactyl ZFs designed using Oligomerized Pool ENgineering (OPEN) and tested for intracellular binding
(Supplementary Fig. 1 and Supplementary Note 1)1,5. To generate two hexadactyl
ZF sensors of high affinity and specificity, we first applied an extended linker that permits finger multimerization without excessive DNA strain, and merged two pairs of the tridactyl ZFs17, 18. The hexadactyl ZFs were fused to VP64 and transformed
into synthetic TFs. Reporters were prepared for each ZF-‐TF encoding GFP
modulated by a single copy of the two original nine bp sites with 0-‐2 unrelated bps between (Supplementary Fig. 2a). We transiently co-‐transfected human
embryonic kidney (HEK 293FT) or HeLa cells with each ZF-‐TF and its reporter, assessing intracellular binding by measuring GFP fluorescence via flow cytometry. The hexadactyl ZF-‐TFs were markedly more active than their tridactyl counterparts (Supplementary Fig. 2b,c). To assess whether the hexadactyl ZFs could overcome potential binding inhibition imposed by intein fusion in a relevant, intracellular context, we attached the IC (C-‐terminal) or IN (N-‐terminal) intein domains, C-‐
terminally mounted VP64, and retested activation (Fig. 2 and Supplementary Fig.
3a,b). Although binding of the hexadactyl ZF-‐intein proteins was encumbered by
intein fusion, the chimers were yet able to achieve a much greater output than the tridactyl-‐intein fusions. We found that a 2× (GGGS)3 linker between the ZF and
intein domains yielded a higher level of GFP activation than the 1× or 3× forms, and used this linker thereafter (Supplementary Fig. 3c-‐e).
ZF9 (Supplementary Fig. 1b,c) was chosen as the eventual split response factor, as it performed well in tested cell lines and its operator yielded low basal expression. GFP was selected as a reporter for response circuit characterization. To improve the dynamic range of GFP activation, ZF9 operators were multimerized
(Supplementary Fig. 4a). The output signal strength increased with the number of sites, basal expression (in ZF9-‐TF’s absence) decreased (resulting in a ~300x fold change using the 6x operator circuit), and there was no cross-‐activation of the response circuit by direct binding of the sensor components (Supplementary Fig.
4b-‐d).
Split extein design, construction, and validation
We hypothesized that an effective way to split the ZF9-‐TF extein would be to separate its DNA recognition and trans-‐activation properties, also allowing for the introduction (and retention) of a splice-‐junction (SJ) that could promote splicing without impairing ZF9-‐TF’s activity19,20. In the SJ design, we adopted several extein
residues that natively flank both sides of the SceVMA intein or from two previously split proteins (Luciferase and TEV protease), reasoning that the intein might successfully ligate the ZFs and VP64 within this context (Fig. 3a and
Supplementary Fig. 5a)12,21.
First, we ensured that the retained SJs did not interfere with ZF9-‐TF’s ability to activate GFP expression by expressing the ZF-‐TF variants in their post-‐splice forms (Supplementary Fig. 5b). Next, we devised a cis-‐splicing, test platform to gauge whether the intein could splice the split extein and produce a functional TF.
were directly linked to one another via a flexible linker (Fig. 3b)22. Results
indicated that all three variants underwent successful cis-‐splicing, resulting in GFP activation (Fig. 3c and Supplementary Fig. 5c). To confirm that cis-‐splicing yielded a product of anticipated size, protein was isolated and subjected to immuno blotting. FLAG-‐ and HIS-‐tag probing of the N-‐ and C-‐termini revealed a product that co-‐
migrated with the ZF9-‐TF positive control for each of the variants but not for three splice-‐null mutants, which displayed greater accumulation of unspliced precursor (Fig. 3d and Supplementary Fig. 5d-‐f)22. Semi-‐quantitative analysis, by
comparison of splice-‐product signal intensity to that of increasing amounts (volumes) of the ZF9-‐TF positive control, displayed >20% cis-‐splice efficiency (Supplementary Fig. 5g).
Intracellular DNA detection and trans-‐splicing
To form the complete sensors we fused the three SJ variants of the split-‐extein with their hexadactyl sensing components. Cells were transfected with a plasmid
encoding the sensor pair, a plasmid carrying the target sequences, and one containing the response circuit (Fig. 4a). Flow cytometry analysis showed substantial fluorescence output for all three variants, indicating target sequence detection (Fig. 4b and Supplementary Fig. 6a). Fluorescence measured in the absence of target sequence, the N-‐ or C-‐terminal sensor, or when a C-‐terminal deletion mutant (lacking the IC domain) was tested, was comparably low, suggesting
extremely low levels of spontaneous trans-‐splicing, confirming earlier reports and that both intact intein domains are required in order to generate a response
the greatest activity and was chosen for further characterization. Targets containing zero, four, eight, or twelve bp gaps (nonrelated bps) between the two 18 bp binding sites produced response signals, with the contiguous 36 bp site displaying the highest output (Supplementary Fig. 6c). Increasing amounts (nanograms) of target plasmid resulted in a dose-‐response effect correlating between plasmid amount and signal strength (Fig. 4c).
Sensor binding strength is essential to overcome off-‐target dilution but could possibly limit the interchange of the post-‐ and pre-‐splice sensors upon the DNA. We reasoned that by moderately lowering affinity but not specificity, interchange might be improved. Eight RàA mutations expected to lower ZF binding affinity were introduced into the ZF backbones and output signals of wildtype and mutant sensor pairs were compared23. Equal-‐molar target plasmids containing a gradient of target
sequence instances (0-‐4, 8) produced a correlative effect for wildtype and mutant sensors but the output of the latter was higher, suggesting improved interchange (Fig. 4d). We also assessed the difference between a non-‐replicative and replicative target and found the signal to be substantially higher in the latter case
(Supplementary Note 2 and Supplementary Fig. 7a,b). To demonstrate the platform’s extensibility, a second sensor pair was constructed (ZF3/ZF2 and ZF4/ZF5) and co-‐transfected with either its own target or that of the original sensors. Each pair detected its specific sequence while ignoring the other’s, highlighting the platform’s robustness and orthogonality (Fig. 4e).
Gene-‐directed enzyme prodrug therapy (GDEPT) involves the metabolism of a nontoxic prodrug into a cytotoxic form24. One example is the NTR+CB 1954 pair,
wherein the toxicity of CB 1954 (5-‐(aziridin-‐1-‐yl)-‐2,4-‐dinitrobenzamide) is dependent upon its reduction by the bacterial nitroreductase, NTR, which transforms it into an agent of DNA interstrand crosslinking and apoptosis25.
Diffusion into neighboring cells leads to increased potency through a “bystander” effect. This dual-‐component method was used to test the system’s ability to link sequence recognition with triggered apoptosis. With GFP replaced by NTR in the response circuit and samples grown in prodrug-‐supplemented media, NTR
expression and the ensuing CB 1954 reduction should, in principle, only occur in the presence of the target sequence26.
HEK 293FT cells were plated in media containing a range of prodrug concentrations and transfected (at 50% rate, based on a control) with either the sensor system containing an “empty” response circuit or the full system, in the presence or absence of target sequences (Fig. 5a and Supplementary Fig. 8a). Apoptosis was measured using flow cytometry after 48 h and 72 h via Annexin V-‐ FITC and propidium iodide (PI) staining. Cells with the empty response circuit were not notably affected by the prodrug, and cells transfected with the full system but no target, were only moderately affected. However, target detection led to markedly elevated levels of apoptotic cells displaying a clear trend in line with the drug gradient. Microscopy revealed that at 96 h, control populations were excessively confluent (Fig. 5b and Supplementary Fig. 8b). Conversely, target-‐positive samples at 16 or 32 (μM) displayed severe or complete cell death, providing evidence of
sequence-‐recognition and a bystander effect. To demonstrate a dual-‐output response, a GFP-‐IRES-‐NTR construct was also generated and simultaneously showed target reporting and apoptosis (Supplementary Fig. 8c).
Virus detection in infected mammalian cells
To test the system’s capability of detecting viral infection, we adopted adenovirus type 5 (Ad5) as a model27. Virus was generated with constitutive expression of blue
fluorescent protein (BFP) to track infected cells. Two sub-‐strains were prepared in which target sequences of sensor pair 1 or 2 (T1, T2) were embedded into the viral genome adjacent to the packaging signal (ψ) (Fig. 6a). Replication-‐permissive HEK 293FT cells were transfected with either of the sensor pairs, followed by infection with virus containing T1, T2, or neither target. Results revealed that both sensor pairs solely detected virus carrying their specific targets (Fig. 6b,c). Finally, the expression, reporting, and marker elements were condensed into a single construct. Only cells receiving both the detection system and virus displayed a substantial GFP response (Supplementary Fig. 9). Together, these studies demonstrate the
feasibility of using this system for sequence-‐specific DNA detection in mammalian cells.
Discussion
Designable DNA-‐binding proteins have been developed to regulate gene expression in endogenous pathways and insulated exogenous gene networks. Using such proteins, we have developed a system that is sufficiently robust so as to facilitate sequence detection within mammalian cells but also highly modular in order to
allow its application to a range of scientific settings, including synthetic biology, diagnostics, and basic research. Below, we discuss the system’s underlying features as well as possible expansions through which the system could be applied.
The recognition stage may be adjusted to detect any target sequence by installing the appropriate ZF component. Also, alternative DNA-‐binding
technologies could be tested, including TALEs or the CRISPR/Cas system28. Another
option would be the use of designer RNA-‐binding proteins for the detection of pathogens with RNA-‐based genomes29. Cells infected with a particular pathogen,
retaining viral latency or undergoing carcinogenesis could be culled through the expression of pro-‐apoptotic proteins, cell death-‐activating surface molecules, or cytokines for immune system recruitment 30,31,32, 33.
This system has the potential to address many research challenges. For example, it could be adapted to identify or rescue only cells that have received a construct during transfection or transduction, using sensors programmed to
recognize the vector backbone sequence. Sensors could be standardized, integrated into stable cell lines, and paired with specific delivery vectors, streamlining the delivery process and preserving packaging space.
Another possible use of this platform regards the CRISPR-‐Cas system, which is commonly utilized to introduce mutations or replace genomic sequences via homology directed repair8. An intrinsic part of such studies is the identification and
isolation of cells containing the edit. PCR or DNA sequencing can measure
frequencies but cannot be applied to live cells. Furthermore, to establish a clonal cell line containing the edit, multiple lines must undergo screening. Instead, cells could
be equipped with sensors designed to bind within the insertion or deletion site, easily identifying on a single-‐cell basis cells that have undergone the desired editing, showing gain or loss of the report signal. This approach could greatly benefit studies of genome editing, increasing throughput and efficiency.
Similarly, this concept could be used to produce clonal cell lines harboring HIV provirus, which are essential for studying viral latency. Such cell lines are screened through a tedious process that involves infection, serial dilution, and viral activation to identify clones that contain the provirus34. Using a sense-‐and-‐respond
system programmed to recognize the known proviral sequence and produce a reporter signal, allowing for the rapid identification of positive cells and their subsequent characterization, could markedly shorten this process.
This system could also contribute to studies of chromosomal aberrations
leading to cancer and other diseases. In some instances, fluorescence in situ
hybridization may be applied and in others, genomic DNA may be subjected to PCR to indicate the presence of chromosomal inversions35. However, PCR-‐based
methods cannot be used to study real-‐time biology nor disclose the aberration frequency within cell populations. Instead, by designing sensors whose proximity would be dependent on the occurrence of a specific chromosomal aberration, researchers could study the biology of living cells and also measure the impact of genetic predisposition or carcinogens.
The study of whole-‐genome 3D architecture relies on high-‐throughput chromosomal confirmation capture techniques (3C, 4C, 5C, Hi-‐C) to map long-‐range interactions36. Stages include DNA crosslinking, digestion, deep-‐sequencing,
microarrays, and the computational reconstruction of the 3D structure. With appropriate sensor design, a proximity-‐based DNA sense-‐and-‐respond approach could confirm the validity of proposed long-‐range interactions, in living cells, also providing insight about whole-‐genome real-‐time dynamics.
The sensors presented here may also expand the limited range of orthogonal inducible components for synthetic biology researchers, especially in cell-‐free platforms. Although engineered TFs can bind any sequence, providing limitless operators, the lack of inducible parts constrains the level of complexity and tunability synthetic circuits can achieve. Here, each sensor pair corresponds to its own binding site, and numerous pairs can be generated. In the form of short dsDNA duplexes, their binding sites could be used as inducers to control the reconstitution of orthogonal split ZF-‐TFs, yielding highly tunable and layered gene networks.
ACKNOWLEDGMENTS
We are grateful to S. Modi for helpful discussions and critical reviews of the
manuscript. HEK 293FT cells were donated by the R. Weiss lab, MIT. This work was supported by funding from the Defense Advanced Research Projects Agency grant DARPA-‐BAA-‐11-‐23, Defense Threat Reduction Agency grant HDTRA1-‐14-‐1-‐0006, and AFOSR-‐BRI grant FA9550-‐14-‐1-‐0060.
AUTHOR CONTRIBUTIONS
S.S. and J.J.C. conceived the study, analyzed data and wrote the paper. S.S. designed and performed the experiments.
COMPETING FINANICIAL INTERESTS
The authors declare no competing financial interests.
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Figure Legends
Figure 1. A DNA sense-‐and-‐respond system. A toolbox of parts (left) comprising the
system and a schematic (right) of the detection of a specific DNA sequence in the mammalian cell nucleus are shown. Two modular ZF-‐based sensors are
programmed to dock adjacently upon target DNA. Each sensor contains an
artificially split SceVMA intein domain that is fused to the split extein. The extein is a ZF-‐TF, split between the binding and trans-‐activating domains. Target detection facilitates intein co-‐localization followed by extein trans-‐splicing. The spliced ZF-‐TF activates the expression of an interchangeable response gene downstream of its operator in the response circuit.
Figure 2. ZF-‐intein sensor design. Tridactyl and hexadactyl (generated by merging
exteins) and intracellular binding activity was tested. Operators for hexadactyl proteins consisted of two contiguous nine bp sites (0gap) or two nine bp sites interrupted with one (1gap) or two (2gap) unrelated bps, with GFP as a reporter. Mean GFP fluorescence (blue bars) of biological duplicate samples is displayed as overlaid bars and compared to control samples in the absence of ZF-‐TFs (grey bars).
Figure 3. Split extein and splice-‐junction (SJ) design and cis-‐splice validation. (a)
Schematic: Extein residues from split luciferase introduced into ZF9-‐TF between its ZF and VP64 domains form splice junctions (SJ). Successful extein ligation would result in the ZF9-‐TF-‐V2 version (see also Supplementary Fig. 5). (b) Schematic: Split extein cis-‐splicing was tested in a condition-‐free manner by tethering the divided intein domains to one another with a flexible linker, applied to three extein variants (CIS V1-‐3), and measured through GFP trans-‐activation by the splice product, shown in (c) and Supplementary Fig. 5c. Biological duplicate samples are displayed as overlaid bars. (d) A α-‐HIS-‐probed immuno blot of proteins extracted from HEK 293FT cells expressing the three variants and splice-‐null, double mutants (CIS-‐V1-‐3m), (see also Supplementary Fig. 5d,e,f,g for mutation schematic,
uncropped blot version of (d), α-‐FLAG probing, and quantification). Lane 1 is a ZF9-‐ TF-‐V2 size control distinguishing between unspliced precursor and splice product. Total protein was normalized with α-‐Tubulin.
Figure 4. Intracellular DNA sense-‐and-‐respond. (a) The three components of the
plasmid carrying 0-‐8 instances of the 36 bp target sequence, (iii) response circuit modulated by spliced ZF9-‐TF. (b) Transfection of three sensor pair variants (V1, 2, 3) with (blue) and without (grey) target plasmid (see also Supplementary Fig.
6a,b). (c) Increasing amounts (ng) of transfected target plasmid yield a dose-‐
response output. N-‐terminal sensor is absent in column 1. (d) Signal comparison between wildtype (wt) and mutant sensors using a series of target plasmids
containing 0-‐4, 8 target instances. (e) Orthogonality testing for two sensor pairs and their target plasmids (T1, T2). Mean GFP fluorescence of biological triplicate
samples (for b,c,e) and duplicate samples (for d) are displayed as overlaid bars.
Figure 5. Sequence recognition-‐induced apoptosis. (a) Schematic: target sequence
recognition leads to NTR expression, CB 1954 reduction, and apoptosis.
Transfection (50% rate, see Supplementary Fig. 8a, right) of sensor system with target (blue), without target (grey), or in the absence of the NTR response circuit (orange). Media contained [CB 1954] (0-‐32 μM). Samples collected after 72 h, stained with Annexin V, and measured via flow cytometry. Numbers represent % of cells staining positive for Annexin V (apoptosis), (see also Supplementary Fig. 8a for 48 h measurement). Biological duplicate samples are displayed as overlaid bars of matching color. (b) Phase-‐contrast microscopy 96 h post-‐treatment for cell population viability (see also Supplementary Fig. 8b for full prodrug range). Scale bar is 50 μM.
Figure 6. Adenovirus detection and reporting. (a) Schematic: Instances of targets 1
or 2 (T1, T2) (or no target) inserted into the Ad5 genome downstream of the
packaging signal (ψ). Constitutive BFP expression serves as a viral infection marker. The detection system (mCherry as a transfection marker), based on sensor pair 1 or 2, is present in the cell nucleus prior to infection. Cells were infected with virus containing T1, T2, or no target (Vir (–T)). Sequence-‐specific detection was measured through GFP trans-‐activation. (b) HEK 293FT cells were transfected and infected as described above. Mean GFP fluorescence in mCherry+/BFP+ cells of biological
duplicate samples is displayed as overlaid bars. (c) Epifluorescence microscopy of cells subjected to transfection à infection (mCherry = transfection, BFP = infection, and GFP = detection). Scale bar is 50 μM.
ONLINE METHODS
OPEN ZF library and split SceVMA intein.
The 10 tridactyl ZFs used in this study were developed using OPEN, as previously described5 (See also Supplementary Note 1 and Supplementary Table 2). In all
screening, VP64 was C-‐terminally fused. The SceVMA intein was simultaneously amplified and divided from gDNA purified from Saccharomyces cerevisiae, as
previously described11. IN and IC consist of residues 1-‐184 and 390-‐454, respectively,
in the full SceVMA sequence. Glycine/serine insolating linkers were introduced by gBlock synthesis (Integrated DNA Technologies (IDT)).
General cloning procedures.
Standard cloning procedures were applied (see also Supplementary Note 3). All PCR amplification stages used Phusion polymerase (NEB), according to the
manufacturer’s protocol. All restriction enzymes used throughout this study were Type II, NEB enzymes. Alkaline phosphatase (CIP) was used to prevent linear plasmid re-‐ligation to reduce background (except during the ligation of annealed oligos, described below). Bacterial selection of NEB 10-‐beta electro-‐competent cells during cloning was performed on 1% LB-‐agar plates with 120 ug/mL ampicillin or 50 ug/mL kanamycin.
Bacterial strains, over-‐expression, and EMSA
For cloning, 10-‐beta electrocompetent E. coli (NEB) cells were used, and for protein over-‐expression, MG1655 Pro was used. ZF-‐intein fusion proteins were expressed using the pZE21 plasmid, allowing for kanamycin selection and induction with 100 ng/ml anhydrotetracycline (aTc). ZF5-‐IC and IN-‐ZF5 fusion proteins were N-‐
terminally FLAG-‐tagged, extracted with B-‐Per II (Thermo Scientific), purified with α-‐ FLAG M2 affinity gel, and eluted with FLAG peptide (Sigma Aldrich). Purified protein was subjected to EMSA using biotinylated ZF5 DNA duplex (IDT), a streptavidin-‐ HRP conjugate (Thermo Scientific, 21130), chemiluminescent substrate (Bio-‐Rad), and read with a Gel Logic system (Carestream Mol. Imaging).
Plasmid construction, oligo annealing and ligation (oligo drop)
The reporter constructs and synthetic response circuit were based on pGL4.26 (Promega). Luciferase was replaced with EGFP. pVITRO1/MCS/Neo (InvivoGen) was used to express ZF-‐TFs and sensor chimers in human cells. pBW121 served as a co-‐transfection marker with mCherry driven by pCAG. It also functioned as a target-‐ carrier plasmid (with and w/o SV40 Ori). All instances in which ZF operator
sequences and sensor target sequences were ligated into reporter plasmid or target-‐ carrying plasmid, respectively, were performed via “oligo drop”: single-‐stranded DNA oligos were designed to contain post-‐cut overhangs once annealed with their complimentary strand, compatible with ligation into a linearized plasmid.
Lyophilized oligos were resuspended in annealing buffer (10mM Tris-‐HCl, pH 7.5, 50mM NaCl), equal volumes of sense and anti-‐sense were mixed at 100°, cooled to 24° over 60 min, diluted and ligated into linearized plasmid. Tandem repeats of operator or target sequences were directionally cloned in cycles, using oligos designed to contain a full 5’ cut site within which would replace the abolished (by design) 5’ ligation site at cloning cycle.
Cell culture, cell lines, and transfection.
The three cell lines used in this study are HeLa (ATCC CCL2), HEK 293FT (donated by the Weiss lab, MIT), and 293AD (Cell Biolabs). HeLa and HEK 293FT were selected for their convenience as transformed cell lines (also HEK 293FT expresses the SV40 large T-‐antigen allowing transient replication of plasmids containing the
SV40 ori). 293AD has enhanced adhering qualities for Ad5 production. Cells were not tested for mycoplasma contamination or authenticated. Cells were cultured in 4.5 g/L glucose, L-‐glutamine DMEM (Corning) supplemented with 10% FBS, penicillin 100 I.U./mL, and streptomycin 100 μg/mL. The standard transfection protocol was as follows, briefly: 100-‐200K cells were plated in 12-‐well format and transfected 24h later with 1-‐1.5 ug combined DNA using polyethylenimine (PEI) as a transfection reagent. 48h post-‐transfection, cells were examined using a Nikon Eclipse Ti epifluorescence microscope, trypsinized, and prepared for flow cytometry analysis.
Mammalian protein extraction and immunoblots.
48h post-‐transfection, total protein was extracted using RIPA (Thermo Scientific) from HEK 293FT cells expressing cis-‐splice test proteins or ZF9-‐TF, tagged with FLAG or 3xHIS at the N-‐ or C-‐ terminus, respectively. Protein was loaded on a Mini-‐ PROTEAN TGX gel, transferred to a 0.45 μm nitrocellulose membrane using a Trans-‐ Blot SD cell and normalized via α-‐Tubulin (abcam). Membranes were probed with either α-‐FLAG-‐M2 (Sigma Aldrich, F3165) or α-‐HIS (GenScript, A00174-‐40) primary antibodies and secondary HRP-‐conjugated antibodies (Bio-‐Rad, #1721011 and #1706515), and imaged with a Gel Logic system (Carestream Mol. Imaging).
Sequence recognition-‐induced apoptosis via CB 1954/NTR. CB 1954 (Sigma
described above and supplemented with 0-‐32 [μM] prodrug. After 48h or 72h, cells were collected, including growth media, and subjected to Annexin V-‐FITC and PI staining according to manufacturer’s instructions (BD Biosciences), followed by flow cytometry. Cells were also analyzed via light microscopy at 96h.
Adenovirus preparation, titration, and infection. Ad5 was prepared using the
RAPAd CMV adenoviral expression system (Cell Biolabs). Briefly, TagBFP was cloned downstream of a CMV promoter as an infection marker. Target sequence (0-‐ 8 copies) for sensor pair 1 or 2 was ligated into the viral genome adjacent to the Ad5 ψ. Crude viral extract was prepared via co-‐transfection of the linearized viral
plasmids into a 293AD line (Cell Biolabs), and after collection, was amplified in the same cells and titrated through serial dilution and plaque counting. For
transfection/infection experiments, HEK 293FT cells were transfected with the sensor system as described above and after 24h, infected with virus at an MOI of 5. 48h later, cells were trypsonized, fixed and subjected to flow cytometry analysis.
Flow cytometry and data analysis. All transfections were measured with a BD
FACSAria II flow cytometer (BD Biosciences). Transfection/infections were measured using a BD Fortessa High Throughput Sampler. Means of fluorescence distributions were calculated with FlowJo. In transfections, GFP arbitrary units were collected from cells gated to mCherry and in transfection/infection experiments, gated to mCherry and BFP fluorescence. All experiments were repeated multiple times and data are displayed either as an average of biological triplicates or
duplicates with standard deviation, or as fold change with error propagation calculations. Due to experimental variation, while data trends may be compared between experiments, absolute values should be compared within single
experiments.
Supplementary Notes
Note 1. Tridactyl ZF screening.
At the onset of this work, we utilized ten tridactyl ZFs designed using OPEN (Oligomerized Pool ENgineering) and screened with bacterial 2-‐hybrid methods. In order to select two members to serve as DNA sensors and another to function as the split extein, the ZFs were fused to VP64 and transformed into synthetic TFs (Supplementary Fig. 1a). Reporters were prepared for each ZF-‐TF encoding GFP modulated by a single copy of the respective nine bp binding site. We transiently co-‐transfected human embryonic kidney (HEK 293FT) or HeLa cells with each ZF-‐TF and its reporter, assessing intracellular binding by
measuring GFP fluorescence via flow cytometry (Supplementary Fig. 1b,c). While a number of ZF-‐TFs showed marked activation, several displayed fluorescence only moderately higher than their negative controls. Also, a purified tridactyl IN-‐ZF fusion
protein subjected to a gel retardation assay could not bind its operator to any detectable measure (Supplementary Fig. 1d). Therefore, hexadactyl ZF proteins were constructed; to both acquire higher affinity and overcome binding hindrance imposed by fusion to the intein domains, as described in the main text (Fig. 2).
Note 2. Non-‐replicative vs. replicative target.
A replicating target-‐carrying plasmid may more accurately represent an active viral infection compared to a non-‐replicative target plasmid. Therefore, to assess whether the system could distinguish between these two options, the Simian vacuolating virus 40
origin of replication (SV40 ori) was inserted into the target plasmid and tested in HEK 293FT cells (capable of replicating DNA containing this sequence). Epifluorescence microscopy revealed successful target recognition as GFP-‐positive cells were visible only in the presence of the target and even markedly more so, in that of its replicative form (Supplementary Fig. 7).
Note 3. Plasmids, oligos, and component sequences
Plasmid sequences have been deposited to addgene.com and accession numbers have been provided in Supplementary Table 1. Additional sequences of individual components, e.g. -‐ ZFs and their operator sites, SceVMA intein domains and the primers used to amplify them, linker sequences merging tridactyl ZFs to create hexadactyl sensors, Glycine/Serine-‐rich insulating linkers distancing the intein domains from the ZF domains, target sites of all gap sizes for both sensor pairs, localization and immuno blot tags, genes encoding fluorescent proteins, gBlocks used to create a second sensor pair, gBlocks to introduce RàA mutations into ZFs, splice junction linkers-‐ can be found in the Supplementary Tables 2-‐4.
Note 4. Equipment and settings.
Images of blots shown in Fig. 3 and Supplementary Fig. 1,5, were acquired using the Gel Logic 6000 Pro system (Carestream Mol. Imaging) with the manufacturer’s image acquisition software (Carestream MI) for chemiluminescent imaging. Microscopy images in Fig. 5,6 and Supplementary Fig. 4,7,8 were acquired with the following equipment: Nikon Eclipse Ti epifluorescence microscope, Nikon Intensilight C-‐HGFIE, Sutter
Instrument Smartshutter controller, Photometrics CoolSnap HQ2, Nikon TI-‐PS 100W, Prior
ProScan II. DAPI, TxRED, FGP(R)BP, ANALY filters. A Plan Fluor 10×/0.30 objective was used for all images, at a pixel dimension of 1392×1040 and resolution of 72 pixels/inch. Images were analyzed using the manufacturer’s NIS-‐Elements software.
Supplementary Tables
Supplementary Table 1
Name Description Resistance Marker Addgene accession number
pVITRO1-‐SS-‐132 ZF1-‐TF + mCherry kan G418 68729 pVITRO1-‐SS-‐115 ZF2-‐TF + mCherry kan G418 68730 pVITRO1-‐SS-‐126 ZF3-‐TF + mCherry kan G418 68731 pVITRO1-‐SS-‐117 ZF4-‐TF + mCherry kan G418 68732 pVITRO1-‐SS-‐127 ZF5-‐TF + mCherry kan G418 68733 pVITRO1-‐SS-‐128 ZF6-‐TF + mCherry kan G418 68734 pVITRO1-‐SS-‐130 ZF7-‐TF + mCherry kan G418 68735 pVITRO1-‐SS-‐131 ZF8-‐TF + mCherry kan G418 68736 pVITRO1-‐SS-‐113 ZF9-‐TF + mCherry kan G418 68737 pVITRO1-‐SS-‐129 ZF10-‐TF + mCherry kan G418 68738 pGL4.26-‐SS-‐140 ZF1 Op -‐ GFP Amp Hygro 68739 pGL4.26-‐SS-‐79 ZF2 Op -‐ GFP Amp Hygro 68740 pGL4.26-‐SS-‐134 ZF3 Op -‐ GFP Amp Hygro 68741 pGL4.26-‐SS-‐80 ZF4 Op -‐ GFP Amp Hygro 68742 pGL4.26-‐SS-‐135 ZF5 Op -‐ GFP Amp Hygro 68743 pGL4.26-‐SS-‐136 ZF6 Op -‐ GFP Amp Hygro 68744 pGL4.26-‐SS-‐138 ZF7 Op -‐ GFP Amp Hygro 68745 pGL4.26-‐SS-‐139 ZF8 Op -‐ GFP Amp Hygro 68746 pGL4.26-‐SS-‐78 ZF9 Op -‐ GFP Amp Hygro 68747 pGL4.26-‐SS-‐137 ZF10 Op -‐ GFP Amp Hygro 68748 pVITRO1-‐SS-‐148 ZF8/ZF7-‐TF + mCherry kan G418 68749 pVITRO1-‐SS-‐149 ZF6/ZF10-‐TF + mCherry kan G418 68750 pGL4.26-‐SS-‐160 ZF10-‐ZF6 Op 0gap -‐ GFP Amp Hygro 68751
